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. 2019 Aug 27;8:e47362. doi: 10.7554/eLife.47362

Figure 6. The anti-leukemia drug indisulam inhibits DCAF15 function.

(A) Proposed model for DCAF15 gain- and loss-of-function phenotypes triggered by indisulam treatment. (B) Dose response of K562 cells expressing the indicated sgRNAs to indisulam. Relative viability measured by ATP content. N = 2 experiments. Mean and standard deviation are shown. (C) Growth of K562 cells treated with 0.1 μM indisulam over 4 days. N = 12 per timepoint. Mean and standard deviation are shown. (D) Western blot for total RBM39 levels in K562 cells after 0.1 μM indisulam treatment for the indicated number of days. (E) Flow cytometry measurements of the indicated cell surface markers in K562 cells after indisulam treatment. Mean and standard deviation are shown. ***p<0.0001, **p=0.0085, *p=0.013, mean significantly different from 1, one sample T test. (F) CD80 expression measured by flow cytometry in indisulam-treated K562 cells expressing the indicated sgRNAs. Mean and standard deviation are shown. N = 4–6 samples. **p=0.0095, *p=0.0286, ns p>0.1, Mann-Whitney test. (G) Effect of indisulam treatment on CD80 expression in the indicated cell lines. N = 3–12 samples. Mean and standard deviation are shown. ***p<0.0001, **p=0.0066, mean significantly different from 1, one sample T test. (H) Kaplan-Meier analysis of overall survival in adult AML patients from TCGA LAML project stratified by DCAF15 expression. ‘DCAF15 high’ and ‘DCAF15 low’ represents patients in top or bottom 50% of DCAF15 expression, respectively. N = 142 patients. 95% confidence interval shown. Median survival, 16.17 vs 12.18 months. P-value from log-rank test. (I) Kaplan-Meier analysis of overall survival in pediatric AML patients from TARGET project stratified by DCAF15 expression. ‘DCAF15 high’ and ‘DCAF15 low’ represents patients in top or bottom 20% of DCAF15 expression, respectively. N = 76 patients. 95% confidence interval shown. Median survival, 21.17 vs NA. P-value from log-rank test.

Figure 6.

Figure 6—figure supplement 1. Additional indisulam experiments.

Figure 6—figure supplement 1.

(A) Relationship between DCAF15 mRNA expression and indisulam sensitivity across a panel of cell lines. (B) Time-course of CD80 expression after indisulam treatment in K562 cells measured by flow cytometry. Blue shading indicates period of CD80 upregulation. N = 2–4 samples per timepoint. Mean and standard deviation are shown. (C) CD80 expression measured by flow cytometry across a panel of cancer cell lines. Dashed line indicates background level of antibody staining, with cell lines in red have expressing above background levels of the protein. CML, chronic myelogenous leukemia. BL, Burkitt’s lymphoma. AML, acute myeloid leukemia. AMoL, acute monocytic leukemia. T-ALL, T-acute lymphoblastic leukemia. MM, multiple myeloma. ALCL, anaplastic large cell lymphoma. (D) Growth of Daudi and KU812 cells treated with 0.1 μM indisulam over 4 days. N = 3 per timepoint. Mean and standard deviation are shown. (E) Fold change in CD80 expression in the indicated cell lines after 4 days treatment with 0.1 μM indisulam. (F) Box and whisker plot of DCAF15 expression in TCGA LAML samples, stratified by subtype using the French American British (FAB) AML classification scheme. Analysis from UALCAN portal (Chandrashekar et al., 2017). Subtypes with significantly different median expression values are indicated. (G) FAB classification of adult AML patients in the TCGA LAML project, stratified by top 50% (‘DCAF15 high’) or bottom 50% (‘DCAF15 low’) of DCAF15 expression.